This proposal is designed to develop a hemophilia B nucleic-acid therapy capable of mediating multi-year expression of Factor IX (FIX) at therapeutic levels from a single administration that is anticipated to cost less than 2 years of traditional repeated administration of recombinant FIX. Our proposal benefits greatly from the findings of a previous independent human clinical trial of gene therapy for hemophilia B using similar adeno- associated virus- (AAV-) serotype-2 vector-based delivery of FIX targeting liver. This trial demonstrated the relative safety of the capsid, or vector shell, in not eliciting symptomatic toxicities at doses higher than proposed herein. The study achieved therapeutic levels (11 - 15%) of circulating FIX protein (even 6% of normal activity converts phenotypically severe to mild bleeding disorders) with the highest tested dose offered promise. However, the effect was transient as an apparent immune response gradually eliminated transduced cells. Importantly, 5- and 25-fold lower doses of AAV2 FIX demonstrated more durable transduction, though without evidence for detectable circulating factor. The apparent inability to attain expression in a therapeutic window without triggering an immune-mediated loss of efficacy has hindered the further development of this previous approach. AskBio's proprietary advances in AAV-mediated gene therapy offer the promise to effectively shift the dose-response relation so that the therapeutic window no longer requires a viral load associated with triggering a CTL response. Specifically, feasibility studies demonstrate improved expression from our incorporation of a novel genomic structure, self-complementary adeno-associated virus (scAAV) in contrast to the traditional single-strand AAV used in previous human clinical trials. These scAAV vectors significantly minimize the vector load required to achieve sustained transgene expression in mice. Using scAAV2 with an optimized FIX codon sequence under a liver-specific promoter (scAAV2-hFIX), we have shown correction of hemophilic mice using 15- to 20-fold lower viral vector doses. Our preliminary preclinical studies demonstrate therapeutic levels of circulating FIX at doses comparable to the lowest investigated clinical doses shown to be well tolerated in the previous clinical trial. This proposal builds upon both extensive preclinical feasibility data as well as previous clinical experience to outline critical preclinical and clinical studies to support the commercialization of this first-in-class approach designed to meet the significant medical needs of Hemophilia B patients. Successful completion of the research aims proposed will support advancement into Phase II/III testing in collaboration with a commercialization partner. PUBLIC HEALTH RELEVANCE: This proposal intends to advance Asklepios Biopharmaceutical's development of a gene therapy for Hemophilia B. Designed to generate stable long-term production of blood-coagulation factor-IX levels without the need for re-administration, this therapeutic gene replacement offers the potential to durably normalize blood-coagulation in affected individuals. Reduced patient burden, non-compliance, and exposure to blood products are expected to correlate with reductions in morbidity and mortality - as well as a vastly improved quality of life for Hemophilia B patients in the U.S. alone.